Mass analysis was performed using an EASY-nLCTM 1200 system connected to a Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer (ThermoFisher) equipped with a nano spray interface (New Objective). Peptide mixtures were loaded onto a 75-μm ID, 25 cm length PepMap C18 column (ThermoFisher) packed with 2 μm particles, pore size −100 Å and separated using a segmented gradient in 90 min from 5 to 45% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. Solvent A was 0.1% formic acid in water. The mass spectrometer was operated in the data-dependent mode. Briefly, survey scans of peptide precursors from 350 to 1600 m/z were performed at 120 K resolution with a 2 × 105 ion count target. Fragmentation (MS2) spectra were acquired in the Orbitrap at 60 K resolution. Precursor ions with 3–8 positive charges were selected for fragmentation with dynamic exclusion of 30 secs and isolation window of 1.6 Th. Additional MS2 settings included an automatic gain control target of 50,000 ions and a maximum injection time of 120 ms. The normalized collision energy was set to 30% for HCD scans. MS1 and MS2 scans were acquired in the Orbitrap.
Thermo orbitrap fusion lumos tribrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer is an advanced analytical instrument that combines multiple mass analyzers, including the Orbitrap, to provide high-resolution, accurate-mass (HRAM) measurements of a wide range of analytes. It is capable of performing tandem mass spectrometry (MS/MS) experiments for detailed structural characterization of molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pepmap c18 column, by Thermo Fisher Scientific (2 mentions)
Nano spray interface, by Thermo Fisher Scientific (2 mentions)
Trypsin gold ms grade, by Promega (1 mentions)
C18 column, by Shimadzu (1 mentions)
Dpx 600 spectrometer, by Bruker (1 mentions)
P 2000, by Jasco (1 mentions)
Silica gel, by Shanghai Titan Scientific (1 mentions)
4 protocols using thermo orbitrap fusion lumos tribrid mass spectrometer
1
Orbitrap Lumos-based Proteomics Analysis
2
Proteomic Analysis of SMC Enzyme Extract
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A total of 100 mL of SMC enzyme extract was concentrated using a 10K MWCO Centrifugal Device (Pall, Houston, TX); furthermore, 24 μL of concentrated samples were applied to SDS-PAGE (12.5%). Protein in-gel digestion was performed using MS-grade Trypsin Gold (Promega, Madison, WI) overnight at 37 °C. Tryptic digests were extracted using 10 μL Milli-Q water, followed by extraction with 20 μL 50% acetonitrile/0.1% trifluoroacetic acid. The extracts were dried in a vacuum concentrator at room temperature, dissolved in 1 μL of 5% acetonitrile/0.5% trifluoroacetic acid, and analyzed using a Thermo Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The Proteome DiscovererTM Version 2.2 software (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and Uniprot Fungus database (11,680,304 sequences, 2021.02.01.) were used for protein identification.
3
Comprehensive Analytical Techniques for Compound Characterization
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HR-ESI-MS spectra were selected on a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were used in a Bruker DPX-600 spectrometer (Bruker Company Ltd, Karlsruhe, Germany) with TMS as an internal standard. Optical rotation was measured on the Jasco P-2000 digital polarizer. Preparative HPLC was measured by LC-20AR pump and RID-20A detector (flow rate: 5 mL/min) by a C18 column (5 μm, 20 × 250 mm, 5 mL/min, Shimadzu, Japan). Column chromatography was carried out on silica gel (80–100 mesh and 200–300 mesh, Shanghai Titan Scientific Co, Shanghai, China) and ODS (Octadecylsilyl) (YMC Company Ltd., Kyoto, Japan).
4
Mass Spectrometry Proteomics Workflow
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Mass analysis was performed using an EASY-nLCTM 1200 system connected to a Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher) equipped with a nano spray interface (New Objective). Peptide mixtures were loaded onto a 75-μm ID, 25 cm length PepMap C18 column (Thermo Fisher) packed with 2 μm particles, pore size- 100 Å and were separated using a segmented gradient in 90 min from 5 to 45% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. Solvent A was 0.1% formic acid in water. The mass spectrometer was operated in the data-dependent mode. Briefly, survey scans of peptide precursors from 350 to 1600 m/z were performed at 60 K resolution with a 2 × 105 ion count target. MS data acquisition methods were used CID-MS2/EThcD-MS2/HCD-MS3 for DSSO cross-linked samples. Precursor ions with 3–8 positive charges were selected for CID-MS2/EThcD-MS2 fragmentation with dynamic exclusion of 30 secs and isolation window of 1.6 Th. Fragmentation (MS2) spectra were acquired in the Orbitrap at 30 K resolution. Subsequently, mass-difference-dependent HCD-MS3 acquisitions were triggered if a unique mass difference (Δ = 31.9721 Da) was observed in the CID-MS2 spectrum. The normalized collision energy was set to 25% for CID-MS2 scans and 30% for HCD-MS3 scans. MS1 and MS2 scans were acquired in the Orbitrap whereas MS3 scans were detected in the ion trap.
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